Antisense oligonucleotides which combat aberrant splicing and methods of using the same

ABSTRACT

A method of combatting aberrant splicing in a pre-mRNA molecule containing a mutation is disclosed. When present in the pre-mRNA, the mutation causes the pre-mRNA to splice incorrectly and produce an aberrant mRNA or mRNA fragment different from the mRNA ordinarily encoded by the pre-mRNA. The method comprises hybridizing an antisense oligonucleotide to the pre-mRNA molecule to create a duplex molecule under conditions which permit splicing. The antisense oligonucleotide is one which does not activate RNase H, and is selected to block a member of the aberrant set of splice elements created by the mutation so that the native intron is removed by splicing and the first mRNA molecule encoding a native protein is produced. Oligonucleotides useful for carrying out the method are also disclosed.

This invention was made with government support under Grant No. GM32994from the National Institutes of Health. The Government has certainrights to this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending prior file wrappercontinuation application Ser. No. 08/379,079, filed Jan. 26, 1995, whichis a continuation of abandoned parent case Ser. No. 08/062,471 filed May11, 1993 the disclosure of which is incorporated by reference herein inits entirety.

FIELD OF THE INVENTION

The present invention relates to methods of combating aberrant splicingof pre-mRNA molecules and upregulating gene expression with antisenseoligonucleotides, and antisense oligonucleotides useful for carrying outthe same.

BACKGROUND OF THE INVENTION

The potential of oligonucleotides as modulators of gene expression iscurrently under intense investigation. Most of the efforts are focusedon inhibiting the expression of targeted genes such as oncogenes orviral genes. The oligonucleotides are directed either against RNA(antisense oligonucleotides) (M. Ghosh and J. Cohen, Prog. Nucleic AcidRes. Mol. Biol. 42, 79 (1992); L. Neckers et al., Crit. Rev. Oncog. 3,175 (1992)) or against DNA where they form triplex structures inhibitingtranscription by RNA polymerase II (J. Hanvey et al., Science 258, 1481(1992); W. McShan et al., J. Biol. Chem. 267, 5712 (1992); M. Grigorievet al., J. Biol. Chem. 267, 3389 (1992); G. Duval-Valentin et al., Proc.Natl. Acad. Sci. USA 89, 504 (1992)). To achieve a desired effect theoligonucleotides must promote a decay of the preexisting, undesirableprotein by effectively preventing its formation de novo. Such techniquesare not useful where the object is to upregulate production of thenative protein. Yet, in cases where the expression of a gene isdownregulated because of mutations therein, a means for upregulatinggene expression through antisense technology would be extremely useful.

SUMMARY OF THE INVENTION

The present invention provides means for using antisenseoligonucleotides to upregulate expression of a DNA containing a mutationwhich would otherwise lead to downregulation of that gene by aberrantsplicing of the pre-mRNA it encodes.

Accordingly, a first aspect of the present invention is a method ofcombatting aberrant splicing in a pre-mRNA molecule containing amutation. When present in the pre-mRNA, the mutation causes the pre-mRNAto splice incorrectly and produce an aberrant mRNA or mRNA fragmentdifferent from the mRNA ordinarily resulting from the pre-mRNA. Moreparticularly, the pre-mRNA molecule contains: (i) a first set of spliceelements defining a native intron which is removed by splicing when themutation is absent to produce a first mRNA molecule encoding a nativeprotein, and (ii) a second set of splice elements induced by themutation which define an aberrant intron different from the nativeintron, which aberrant intron is removed by splicing when the mutationis present to produce an aberrant second mRNA molecule different fromthe first mRNA molecule. The method comprises hybridizing an antisenseoligonucleotide to the pre-mRNA molecule to create a duplex moleculeunder conditions which permit splicing. The antisense oligonucleotide isone which does not activate RNase H, and is selected to block a memberof the aberrant second set of splice elements so that the native intronis removed by splicing and the first mRNA molecule encoding a nativeprotein is produced.

A second aspect of the present invention is a method of upregulatingexpression of a native protein in a cell containing a DNA encoding thenative protein. Which DNA further contains a mutation which causesdownregulation of the native protein by aberrant splicing thereof. Moreparticularly, the DNA encodes a pre-mRNA, the pre-mRNA having thecharacteristics set forth above. The method comprises administering tothe cell an antisense oligonucleotide having the characteristicsdescribed above so that the native intron is removed by splicing and thenative protein is produced by the cell.

A third aspect of the present invention is an antisense oligonucleotideuseful for combatting aberrant splicing in a pre-mRNA moleculecontaining a mutation. The pre-mRNA molecule contains a first set andsecond set of splice elements having the characteristics set forthabove. The antisense oligonucleotide comprises an oligonucleotide which(i) hybridizes to the pre-mRNA to form a duplex molecule; (ii) does notactivate RNase H; and (iii) blocks a member of the aberrant second setof splice elements.

The foregoing and other objects and aspects of the present invention arediscussed in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of pre-mRNAs. Boxes indicate exons; heavylines, introns. Positions of the mutations (110 and 705) relative tonucleotide 1 of IVS 1 and IVS 2, respectively are shown above the HBΔ6clone. Numbers below indicate the length, in nucleotides, of exons andintrons. Antisense oligonucleotides are indicated by the numbered shortbars below β¹¹⁰ and IVS2⁷⁰⁵ constructs, and splicing pathways by thedashed lines.

FIG. 2 shows the reversal of aberrant splicing by oligonucleotide 1directed against the normal branch point in intron 1 of β-globinpre-mRNA. The structure of the products and intermediates is depicted onthe right; their size in nucleotides is shown on the left. An asteriskdenotes aberrant mobility of lariat-containing intermediates. The samedesignations are used in the subsequent figures. Lane 1 shows splicingof control HBΔ6 pre-mRNA; lane 2 shows splicing of β¹¹⁰ pre-mRNA; lanes3-8 show splicing of β¹¹⁰ pre-mRNA in the presence of increasing amounts(indicated at the top of the figure) of oligonucleotide 1; lane 9 showssplicing of β¹¹⁰ pre-mRNA in the presence of oligonucleotide 3, targetedto a sequence in intron 2 of β-globin pre-mRNA.

FIG. 3 shows the effects of oligonucleotide 2, directed against theaberrant 3' splice site in intron 1 of β¹¹⁰ pre-mRNA. Lane 1 showssplicing of β¹¹⁰ pre-mRNA; lanes 2-7 show splicing of β¹¹⁰ pre-mRNA inthe presence of increasing amounts (indicated at the top of the figure)of oligonucleotide 2.

FIG. 4 shows the reversal of aberrant splicing of IVS2⁷⁰⁵ pre-mRNA byoligonucleotide 3 directed against the cryptic 3' splice site andoligonucleotide 4 directed against the aberrant 5' splice site in intron2 of the IVS2⁷⁰⁵ pre-mRNA. Lane 1 shows input RNA; lanes 2 and 3 showsplicing of control transcripts (indicated at the top of the figure);lanes 4-8 and 9-13 show splicing of IVS2⁷⁰⁵ pre-mRNA in the presence ofoligonucleotide 3 and oligonucleotide 4, respectively. The amounts ofthe oligonucleotides in the reaction are indicated at the top. "?" onthe left indicates apparent degradation product.

DETAILED DESCRIPTION OF THE INVENTION

Introns are portions of eukaryotic DNA which intervene between thecoding portions, or "exons," of that DNA. Introns and exons aretranscribed into RNA termed "primary transcript, precursor to mRNA" (or"pre-mRNA"). Introns must be removed from the pre-mRNA so that thenative protein encoded by the exons can be produced (the term "nativeprotein" as used herein refers to naturally occuring, wild type, orfunctional protein). The removal of introns from pre-mRNA and subsequentjoining of the exons is carried out in the splicing process.

The splicing process is actually a series of reactions, mediated bysplicing factors, which is carried out on RNA after transcription butbefore translation. Thus, a "pre-mRNA" is an RNA which contains bothexons and intron(s), and an "mRNA" is an RNA in which the intron(s) havebeen removed and the exons joined together sequentially so that theprotein may be translated therefrom by the ribosomes.

Introns are defined by a set of "splice elements" which are relativelyshort, conserved RNA segments which bind the various splicing factorswhich carry out the splicing reactions. Thus, each intron is defined bya 5' splice site, a 3' splice site, and a branch point situatedtherebetween. These splice elements are "blocked", as discussed herein,when an antisense oligonucleotide either fully or partially overlaps theelement, or binds to the pre-mRNA at a position sufficiently close tothe element to disrupt the binding and function of the splicing factorswhich would ordinarily mediate the particular splicing reaction whichoccurs at that element (e.g., binds to the pre-mRNA at a position within3, 6, or 9 nucleotides of the element to be blocked).

The mutation in the native DNA and pre-mRNA may be either a substitutionmutation or a deletion mutation which creates a new, aberrant, spliceelement. The aberrant splice element is thus one member of a set ofaberrant splice elements which define an aberrant intron. The remainingmembers of the aberrant set of splice elements may also be members ofthe set of splice elements which define the native intron. For example,if the mutation creates a new, aberrant 3' splice site which is bothupstream from (i.e., 5' to) the native 3' splice site and downstreamfrom (i.e., 3' to) the native branch point, then the native 5' splicesite and the native branch point may serve as members of both the nativeset of splice elements and the aberrant set of splice elements. In othersituations, the mutation may cause native regions of the RNA which arenormally dormant, or play no role as splicing elements, to becomeactivated and serve as splicing elements. Such elements are referred toas "cryptic" elements. For example, if the mutation creates a newaberrant mutated 3' splice site which is situated between the native 3'splice site and the native branch point, it may activate a crypticbranch point between the aberrant mutated 3' splice site and the nativebranch point. In other situations, a mutation may create an additional,aberrant 5' splice site which is situated between the native branchpoint and the native 5' splice site and may further activate a cryptic3' splice site and a cryptic branch point sequentially upstream from theaberrant mutated 5' splice site. In this situation, the native intronbecomes divided into two aberrant introns, with a new exon situatedtherebetween. Further, in some situations where a native splice element(particularly a branch point) is also a member of the set of aberrantsplice elements, it can be possible to block the native element andactivate a cryptic element (i.e., a cryptic branch point) which willrecruit the remaining members of the native set of splice elements toforce correct splicing over incorrect splicing. Note further that, whena cryptic splice element is activated, it may be situated in either theintron or one of the adjacent exons. Thus, depending on the set ofaberrant splice elements created by the particular mutation, theantisense oligonucleotide may be synthesized to block a variety ofdifferent splice elements to carry out the instant invention: it mayblock a mutated element, a cryptic element, or a native element; it mayblock a 5' splice site, a 3' splice site, or a branch point. In general,it will not block a splice element which also defines the native intron,of course taking into account the situation where blocking a nativesplice element activates a cryptic element which then serves as asurrogate member of the native set of splice elements and participatesin correct splicing, as discussed above.

The length of the antisense oligonucleotide (i.e., the number ofnucleotides therein) is not critical so long as it binds selectively tothe intended location, and can be determined in accordance with routineprocedures. In general, the antisense oligonucleotide will be from 8, 10or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length.

Antisense oligonucleotides which do not activate RNase H can be made inaccordance with known techniques. See, e.g., U.S. Pat. No. 5,149,797 toPederson et al. (The disclosures of all patent references cited hereinare to be incorporated herein by reference). Such antisenseoligonucleotides, which may be deoxyribonucleotide or ribonucleotidesequences, simply contain any structural modification which stericallyhinders or prevents binding of RNase H to a duplex molecule containingthe oligonucleotide as one member thereof, which structural modificationdoes not substantially hinder or disrupt duplex formation. Because theportions of the oligonucleotide involved in duplex formation aresubstantially different from those portions involved in RNase H bindingthereto, numerous antisense oligonucleotides which do not activate RNaseH are available. For example, such antisense oligonucleotides may beoligonucleotides wherein at least one, or all, of the internucleotidebridging phosphate residues are modified phosphates, such as methylphosphonates, methyl phosphonothioates, phosphoromorpholidates,phosphoropiperazidates and phosphoramidates. For example, every otherone of the internucleotide bridging phosphate residues may be modifiedas described. In another non-limiting example, such antisenseoligonucleotides are oligonucleotides wherein at least one, or all, ofthe nucleotides contain a 2' loweralkyl moiety (e.g., C1-C4, linear orbranched, saturated or unsaturated alkyl, such as methyl, ethyl,ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example,every other one of the nucleotides may be modified as described. Seealso P. Furdon et al., Nucleic Acids Res. 17, 9193-9204 (1989); S.Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401-1405 (1990); C.Baker et al., Nucleic Acids Res. 18, 3537-3543 (1990); B. Sproat et al.,Nucleic Acids Res. 17, 3373-3386 (1989); R. Walder and J. Walder, Proc.Natl. Acad. Sci. USA 85, 5011-5015 (1988).

The methods, oligonucleotides and formulations of the present inventionhave a variety of uses. They are useful in any fermentation processwhere it is desired to have a means for downregulating expression of agene to be expressed until a certain time, after which it is desired toupregulate gene expression (e.g., downregulate during the growth phaseof the fermentation and upregulate during the production phase of thefermentation). For such use, the gene to be expressed may be any geneencoding a protein to be produced by fermentation so long as the genecontains a native intron. The gene may then be mutated by any suitablemeans, such as site-specific mutagenesis (see T. Kunkel, U.S. Pat. No.4,873,192) to deliberately create an aberrant second set of spliceelements which define an aberrant intron which substantiallydownregulates expression of the gene. The gene may then be inserted intoa suitable expression vector and the expression vector inserted into ahost cell (e.g., a eukaryotic cell such as a yeast, insect, or mammaliancell (e.g., human, rat)) by standard recombinant techniques. The hostcell is then grown in culture by standard fermentative techniques. Whenit is desired to upregulate expression of the mutated gene, an antisenseoligonucleotide, in a suitable formulation, which binds to a member ofthe aberrant second set of splice elements, is then added to the culturemedium so that expression of the gene is upregulated.

The methods, oligonucleotides and formulations of the present inventionare also useful as in vitro or in vivo tools to examine splicing inhuman or animal genes which are developmentally and/or tissue regulated.Such experiments may be carried out by the procedures describedhereinbelow, or modification thereof which will be apparent to skilledpersons.

The methods, oligonucleotides and formulations of the present inventionare also useful as therapeutic agents in the treatment of diseaseinvolving aberrant splicing, such as β-thalassemia (wherein theoligonucleotide would bind to β-globin, particularly human, pre-mRNA),α-thalassemia (wherein the oligonucleotide would bind to α-globinpre-mRNA), Tay-Sachs syndrome (wherein the oligonucleotide would bind toβ-hexoseaminidase α-subunit pre-mRNA), phenylketonuria (wherein theoligonucleotide would bind to phenylalanine hydroxylase pre-mRNA) andcertain forms of cystic fibrosis (wherein the oligonucleotide would bindthe cystic fibrosis gene pre-mRNA), in which mutations leading toaberrant splicing of pre-mRNA have been identified (See, e.g., S. Akliet al., J. Biol. Chem. 265, 7324 (1990); B. Dworniczak et al., Genomics11, 242 (1991); L-C. Tsui, Trends in Genet. 8, 392 (1992)).

Examples of β-thalassemia which may be treated by the present inventioninclude, but are not limited to, those of the β¹¹⁰, IVS1⁵, IVS1⁶,IVS2⁶⁵⁴, IVS2⁷⁰⁵, and IVS2⁷⁴⁵ mutant class (i.e., wherein the β-globinpre-mRNA carries the aforesaid mutations).

The term "antisense oligonucleotide" includes the physiologically andpharmaceutically acceptable salts thereof: i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto. Examples of such salts are (a)salts formed with cations such as sodium, potassium, NH₄ ⁺, magnesium,calcium, polyamines such as spermine and spermidine, etc.; (b) acidaddition salts formed with inorganic acids, for example hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid andthe like; (c) salts formed with organic acids such as, for example,acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid,fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid,benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamicacid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonicacid, naphthalenedisulfonic acid, polygalacturonic acid, and the like;and (d) salts formed from elemental anions such as chlorine, bromine,and iodine.

Formulations of the present invention comprise the antisenseoligonucleotide in a physiologically or pharmaceutically acceptablecarrier, such as an aqueous carrier. Thus, formulations for use in thepresent invention include, but are not limited to, those suitable forparenteral administration, including subcutaneous, intradermal,intramuscular, intravenous and intraarterial administration, as well astopical administration (i.e., administration of an aerosolizedformulation of respirable particles to the lungs of a patient afflictedwith cystic fibrosis). The formulations may conveniently be presented inunit dosage form and may be prepared by any of the methods well known inthe art. The most suitable route of administration in any given case maydepend upon the subject, the nature and severity of the condition beingtreated, and the particular active compound which is being used.

The present invention provides for the use of antisense oligonucleotideshaving the characteristics set forth above for the preparation of amedicament for upregulating gene expression in a patient afflicted withan aberrant splicing disorder, as discussed above. In the manufacture ofa medicament according to the invention, the antisense oligonucleotideis typically admixed with, inter alia, an acceptable carrier. Thecarrier must, of course, be acceptable in the sense of being compatiblewith any other ingredients in the formulation and must not bedeleterious to the patient. The carrier may be a solid or a liquid. Oneor more antisense oligonucleotides may be incorporated in theformulations of the invention, which may be prepared by any of the wellknown techniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory therapeuticingredients.

Formulations of the present invention may comprise sterile aqueous andnon-aqueous injection solutions of the active compound, whichpreparations are preferably isotonic with the blood of intendedrecipient and essentially pyrogen free. These preparations may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions may include suspending agents andthickening agents. The formulations may be presented in unit dose ormulti-dose containers, for example sealed ampoules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

In the formulation the antisense oligonucleotide may be contained withina lipid particle or vesicle, such as a liposome or microcrystal, whichmay be suitable for parenteral administration. The particles may be ofany suitable structure, such as unilamellar or plurilamellar, so long asthe antisense oligonucleotide is contained therein. Positively chargedlipids such asN-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or"DOTAP," are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. See, e.g., U.S. Pat.Nos. 4,880,635 to Janoff et al.; 4,906,477 to Kurono et al.; 4,911,928to Wallach; 4,917,951 to Wallach; 4,920,016 to Allen et al.;4,921,757 toWheatley et al.; etc.

The dosage of the antisense oligonucleotide administered will dependupon the particular method being carried out, and when it is beingadministered to a subject, will depend on the disease, the condition ofthe subject, the particular formulation, the route of administration,etc. In general, intracellular concentrations of the oligonucleotide offrom 0.05 to 50 μM, or more particularly 0.2 to 5 μM, are desired. Foradministration to a subject such as a human, a dosage of from about0.01, 0.1, or 1 mg/Kg up to 50, 100, or 150 mg/Kg is employed.

The present invention is explained in greater detail in the followingnon-limiting examples. Nucleotide sequences are presented herein bysingle strand only, in the 5' to 3' direction, from left to right.

EXAMPLE 1 Structure and Construction of Pre-mRNAs

The construction and structure of various human β-globin pre-mRNAmolecules is illustrated in FIG. 1. Boxes indicate exons; heavy lines,introns. Positions of the mutations (110 and 705) relative to nucleotide1 of IVS 1 and IVS 2, respectively are shown above the HBΔ6 clone.Numbers below indicate the length, in nucleotides, of exons and introns.Antisense oligonucleotides (discussed in detail below) are indicated bythe numbered short bars below β¹¹⁰ and IVS2⁷⁰⁵ constructs, and splicingpathways by the dashed lines. All pre-mRNAs were transcribed by SP6 RNApolymerase (M. Konarska et al., Cell 38, 731 (1984)) from appropriatefragments of human β-globin gene subcloned into the SP64 vector. HBΔ6(A. Krainer et al., Cell 36, 993 (1984)) contains the whole humanβ-globin gene. The ¹¹⁰ construct contains exons 1 and 2 and wassubcloned from the original thalassemic clone (R. Spritz et al., Proc.Natl. Acad. Sci. USA 78, 2455 (1981)). Before transcription, plasmidswere linearized at the BamHI site. To construct the IVS2⁷⁰⁵ plasmid, afragment of HBΔ6 containing virtually the entire second exon, the entiresecond intron, and a major portion of the third exon was first subclonedin SP64 and subsequently subjected to site specific mutagenesis inaccordance with known techniques (T. Kunkel et al., Methods Enzymol.154, 367 (1987)) to introduce a T to G mutation at nucleotide 705 of theintron. Transcription was then carried out on a plasmid linearized atthe PvuII site.

EXAMPLE 2 Synthesis of Antisense 2'O-methyl-Oligonucleotides

2'-O-methyl-Oligoribonucleotides for use in the examples describedherein were synthesized in accordance with known techniques usingreagents from Glen Research (Sterling, Va.) and purified in accordancewith known techniques using the SUREPURE™ purification kit availablefrom US Biochemicals.

2'-O-methyl-oligoribonucleotides produced were referred to as oligo 1through oligo 5.

Oligo 1 (GUCAGUGCCUAUCA)(SEQ ID NO:1), complementary to nucleotides82-95 of intron 1, is targeted against the normal branch point, andoligo 2 (AUAGACUAAUAGGC)(SEQ ID NO:2), complementary to nucleotides103-116 of intron 1, against the aberrant 3' splice site created by β¹¹⁰mutation in intron 1 of the β-globin gene. Oligo 3 (CAUUAUUGCCCUGAAAG)(SEQ ID NO:3), complementary to nucleotides 573-589 of intron 2, istargeted against the cryptic 3' splice site at nucleotide 579 of thesecond intron and oligo 4 (CCUCUUACCUCAGUUAC) (SEQ ID NO:4),complementary to nucleotides 697-713, is targeted against the aberrant5' splice site created by the mutation at nucleotide 705 in the secondintron of IVS2⁷⁰⁵ pre-mRNA. Oligo 5 (GCUAUUACCUUAACCCAG) (SEQ ID NO:5)is targeted against the aberrant 5' splice site created by the IVS2⁶⁵⁴mutation (nucleotides 643-660 of intron 2). Oligo 6 (GCCUGACCACCAAC)(SEQ ID NO:6) is targeted against the cryptic 5' splice site in exon lofglobin pre-mRNA (nucleotides -23 to -10 relative to nucleotide 1 ofintron 1).

EXAMPLE 3 Reversal of Aberrant Splicing by an Antisense OligonucleotideTargeted Against the Normal Branch Point of Human β-Globin Intron 1

In β¹¹⁰ - thalassemia, a form of the disease predominant in patients ofGreek and Cypriot origin, an A to G mutation at nucleotide 110 of thefirst intron of human β-globin gene creates an additional, aberrant 3'splice site (R. Spritz et al., Proc. Natl. Acad. Sci. USA 78, 2455(1981)). In spite of the presence of the normal 3' splice site, theaberrant site is preferentially used by the splicing machinery,resulting in an incorrectly spliced mRNA that contains 19 nucleotides ofthe intron sequence (FIG. 1). In cells transfected with β¹¹⁰ -globinallele (M. Busslinger et al., Cell 27, 289 (1981); Y. Fukumaki et al.,Cell 28, 585 (1982)) or during splicing of its transcript in nuclearextracts (R. Reed and T. Maniatis, Cell 41, 95 (1985)) (see also FIG. 2,lane 2) correctly spliced mRNA constitutes only about 10% of the splicedproduct, consistent with the markedly reduced levels of normalhemoglobin observed in patients with this form of thalassemia. It wasfound that in β¹¹⁰ pre-mRNA the aberrant 3' splice site recruits thenormal branch point at nucleotide 93 of the intron, competing with thecorrect 3' splice site, and thereby prevents correct splicing (R. Reedand T. Maniatis, Cell 41, 95 (1985)). Significantly for this work,mutations inactivating the normal branch point activate a cryptic branchpoint at nucleotide 107 and result in splicing at the correct 3' splicesite (Y. Zhuang and A. Weiner, Genes and Dev. 3, 1545 (1989)). Aberrantsplicing cannot proceed due to the proximity of the cryptic branch pointto the mutated 3' splice site at position 110.

To test whether antisense oligonucleotides targeted against the normalbranch point sequence would force the splicing machinery to select thecryptic branch point and generate a correctly spliced mRNA, a 14nucleotide long 2'-O-methyl-oligonucleotide (oligonucleotide 1, (SEQ IDNO:1)) was targeted against the branch point sequence in intron 1 ofβ-globin pre-mRNA. The 2'-O-methyl oligonucleotides were selected forthis and subsequent experiments since they are resistant to nucleasesand form stable hybrids with RNA that are not degraded by RNase H (H.Inoue et al., Nucleic Acids Res. 15, 6131 (1987); H. Inoue et al., FEBSLett. 215, 327 (1987); B. Sproat et al., Nucleic Acids Res. 17, 3373(1989)). Degradation by RNase H, seen for example when antisenseoligodeoxynucleotides or their phosphorothioate derivatives are used,would destroy the substrate pre-mRNA and prevent any splicing.

FIG. 2 shows the reversal of aberrant splicing by oligonucleotide 1directed against the normal branch point in intron 1 of β-globinpre-mRNA. Splicing of p³² labeled β¹¹⁰ pre-mRNA (approximately 10⁵ cpmper reaction, 25 fmoles) was carried out in vitro in HeLa cell nuclearextract for 2 hours, essentially as described (A. Krainer et al., Cell36, 993 (1984); Z. Dominski and R. Kole, Mol. Cell. Biol. 12, 2108(1992)) except that the volume of the reaction was doubled to 50 μl.Reaction products were analyzed on an 8% polyacrylamide sequencing geland visualized by autoradiography. The structure of the products andintermediates is depicted on the right, their size in nucleotides isshown on the left. An asterisk denotes aberrant mobility oflariat-containing intermediates. Lane 1, splicing of control HBΔ6pre-mRNA. Lane 2, splicing of β¹¹⁰ pre-mRNA. Lanes 3-8, splicing of β¹¹⁰pre-mRNA in the presence of increasing amounts (indicated at the top ofthe figure) of oligonucleotide 1. Lane 9, splicing of β¹¹⁰ pre-mRNA inthe presence of oligonucleotide 3, targeted to a sequence in intron 2 ofβ-globin pre-mRNA.

Analysis of these data shows that in the control reaction without theoligonucleotide (FIG. 2, lane 2), the ratio of the incorrectly tocorrectly spliced products is approximately 9:1. Addition ofoligonucleotide 1 at concentrations 0.01 to 1.0 μg per reaction (0.05-5μM) causes dose dependent inhibition of aberrant splicing and inductionof the correct splicing of the substrate (FIG. 2, lanes 3-6). At 1.0 μgof the oligonucleotide the ratio of spliced products is reversed to 1:5.The effect of the oligonucleotide is sequence specific since addition of1 μg of an oligonucleotide targeted against the cryptic 3' splice sitein the second intron of the β-globin gene (oligonucleotide 3, (SEQ IDNO:3); see also below) does not affect the original ratio of the splicedproducts (FIG. 2, lane 9). At 2.0 and 4.0 μg of oligonucleotide 1,splicing at both splice sites is inhibited and a 243-mer RNA fragment isgenerated (FIG. 2, lanes 7-8). This fragment accumulates only undersplicing conditions, i.e. in the presence of ATP and other components ofthe splicing mixture, and most likely represents a product of cleavageat the site of the oligonucleotide's binding by an ATP dependentnuclease.

The aberrant 3' splice site generated by the β¹¹⁰ mutation also appearsto be a target for reversal of aberrant splicing by an antisenseoligonucleotide. Blocking of this sequence should be the simplest way offorcing the splicing machinery to use the original 3' splice site at theend of the intron. However, a 14-mer (oligo 2, (SEQ ID NO:2)) directedagainst the aberrant splice site was not effective; at increasingconcentrations of the oligonucleotide accumulation of both splicedproducts was inhibited, the correct one being inhibited somewhat moreefficiently (FIG. 3, lanes 2-5). Splicing was carried out under the sameconditions as described in connection with FIG. 2. Interestingly, thefirst step of the splicing reaction, cleavage at the 5' splice site andformation of the lariat-exon intermediate, seems to be less affected byoligo 2 than the formation of the final spliced product. This is shownby the presence of these intermediates even when 1 or 2 μg of theoligonucleotide were added to the splicing reaction (FIG. 3, lanes 5-6).At 4 μg per reaction cleavage at the 5' splice site is inhibited (FIG.3, lane 7).

The different effects of oligo 1 and oligo 2 reflect complexinteractions among the oligonucleotides, the numerous splicing factorsand sequence elements located in the stretch of 37 nucleotides betweenthe normal branch point and the correct 3' splice site. Clearly,oligonucleotide 1, hybridized to the normal branch point at the 5' endof this region, prevents binding of the splicing factors to thissequence forcing them to select the cryptic branch point downstream.This leads to inhibition of aberrant and induction of correct splicingof β¹¹⁰ pre-mRNA. In contrast, hybridization of oligo 2 to its centrallylocated target sequence may hinder binding of a large number of splicingfactors that assemble in this region and prevent any splicing. Note alsothat this oligonucleotide blocks a significant portion of thepolypyrimidine tract that is essential for splicing to both the aberrantand the correct 3' splice sites. This is an alternative explanation whythis oligonucleotide failed to restore the correct splicing pathway.

EXAMPLE 4 Reversal of Aberrant Splicing by Antisense OligonucleotidesAgainst the 5' and 3' Splice Sites of Human β-Globin Intron 2

Whether an aberrant 3' splice site can nevertheless be used as a targetfor reversal of incorrect splicing was further tested on pre-mRNAcarrying a T to G mutation at position 705 of the second intron of humanβ-globin gene. This mutation (IVS2⁷⁰⁵), found in Mediterraneanthalassemia patients, creates an additional, aberrant 5' splice site 145nucleotides upstream from the normal 3' splice site (C. Dobkin and A.Bank, J. Biol. Chem. 260, 16332 (1985)). During splicing, a cryptic 3'splice site is activated at position 579 of the intron resulting in theremoval of nucleotides 1-578 and 706-850 as separate introns andincorporation of the remaining portion of the intron into the splicedproduct (FIG. 1). In this RNA the distances between each of the sequenceelements involved in splicing exceed 100 nucleotides and no sterichindrance effects by the oligonucleotide should be expected.

The reversal of aberrant splicing of IVS2⁷⁰⁵ pre-mRNA by oligonucleotide3 (SEQ ID NO:3) directed against the cryptic 3' splice site andoligonucleotide 4 (SEQ ID NO:4) directed against the aberrant 5' splicesite in intron 2 of the IVS2⁷⁰⁵ pre-mRNA is shown in FIG. 4. Theconditions of the splicing reaction were the same as described inconnection with FIG. 2 above, except that before use the RNA transcriptwas purified by electrophoresis on a 6% sequencing gel. Lane 1, inputRNA. Lanes 2 and 3, splicing of control transcripts (indicated at thetop of the figure). Lanes 4-8 and 9-13, splicing of IVS2⁷⁰⁵ pre-mRNA inthe presence of oligonucleotide 3 and oligonucleotide 4, respectively.The amounts of the oligonucleotides in the reaction are indicated at thetop. "?" on the left indicates apparent degradation product.

The control transcript containing the second intron of normal β-globinpre-mRNA is spliced efficiently (FIG. 4, lane 2) generating the expectedintermediates (the 5' exon and the large lariats) and the correctlyspliced product, 451 nucleotides in length. Splicing of IVS2⁷⁰⁵ pre-mRNAis also efficient and yields an additional spliced product 577nucleotide long and an expected 348-mer intermediate, resulting from theaberrant splicing pathway caused by the mutation (FIG. 4, lane 3). The1:2 ratio of correctly to incorrectly spliced RNAs is similar to thatobserved previously in vivo. Oligonucleotide 3 (FIG. 1) targeted at theactivated cryptic 3' splice site at nucleotide 579 is very active,inducing dose dependent reversal of splicing to the correct splicingpathway (FIG. 4, lanes 4-8). At 0.1 and 0.4 μg of the oligonucleotideper reaction the reversal is virtually complete. Correct splicing isalso obtained at similar concentrations of oligonucleotide 4 (FIG. 1)targeted against the aberrant 5' splice site created by the mutation atnucleotide 705 of the second intron (FIG. 4, lanes 9-13). At 1 and 2 μgper reaction, either oligonucleotide had no additional effects; at 4 μgper reaction (20 μM) all splicing is inhibited (not shown). Additionalbands, including a strong band marked by "?" in a figure are most likelydue to nuclease degradation of the long (1301 nucleotides) pre-mRNA.

These results show that the cryptic 3' splice site as well as themutated 5' splice site provide suitable targets for specific reversal ofaberrant splicing. Similar effects of oligonucleotides 3 and 4 suggestthat there are no major differences in their accessibilities to thetarget splice sites. Both oligonucleotides are approximately 10 timesmore effective than oligonucleotide 1 used in the experiments shown inFIG. 2. This higher efficiency may be due to several factors.Oligonucleotides 3 and 4 are three nucleotides longer thanoligonucleotide 1 and may form more stable hybrids with RNA. They blockaberrant splice sites, allowing the splicing machinery to use thecorrect splice sites and, presumably, the correct branch point. Incontrast, in β¹¹⁰ pre-mRNA oligonucleotide 1 forces the splicingmachinery to use a suboptimal cryptic branch point sequence, which mayresult in relatively inefficient generation of correctly spliced mRNA.In experiments shown in FIG. 4 the long input pre-mRNA is barelydetectable after 2 hours of the reaction, suggesting its instability.Thus, although the molar concentrations of the oligonucleotides wereessentially the same as in previous experiments they may have been ingreater excess over the substrate pre-mRNA.

In the experiments presented above the oligonucleotides were addedsimultaneously with the other components of the splicing reaction.Prehybridization of the oligonucleotides with the pre-mRNA did notincrease their efficiency and oligonucleotides added 15 minutes afterthe start of the reaction, i.e. after splicing complexes had a chance toform (B. Ruskin and M. Green, Cell 43, 131 (1985)), were almost aseffective (data not shown). These results indicate that oligonucleotidescontaining the 2'-O-methyl modification are able to compete effectivelyfor their target sequences with the splicing factors. The high activityof these compounds is most likely due to their strong hybridization toRNA.

EXAMPLE 5 Reversal of Aberrant Splicing With an AntisenseOligonucleotide Which Blocks the Cryptic 3' Splice Site the IVS1-5 andIVS1-6

This experiment is carried out essentially as described above, exceptthat the thalassemic mutations are the IVS1-5 and IVS1-6 mutations, inwhich the authentic 5' splice site of IVS1 is mutated. Aberrant splicingresulting in thalassemia is apparently due to the fact that mutationsIVS1-5 and IVS1-6 weaken the 5' splice site and allow the cryptic splicesite located 16 nucleotides upstream to successfully compete for thesplicing factors. In this experiement we test whether an oligonucleotideantisense to the cryptic splice site may revert aberrant splicing backto the mutated 5' splice site and restore correct splicing in spite ofthe mutations, since splice sites similar to the mutated ones appearfunctional in other pre-mRNAs. The oligonucleotide employed is oligo 6(SEQ ID NO:6), a 2-O-methyl-ribooligonucleotide produced as described inExample 2 above.

EXAMPLE 6 Reversal of Aberrant Splicing With an AntisenseOligonucleotide Which Blocks the Aberrant 5' Splice Site of the IVS2⁶⁵⁴Mutation

These experiments are carried out essentially as described above, exceptthat the human β-globin pre-mRNA containing the IVS2⁶⁵⁴ mutation isemployed, and oligo 5 (SEQ ID NO:5) is employed.

The IVS2⁶⁵⁴ mutation, frequently identified in thalassemic individualsof Chinese origin, affects splicing by creating an additional 5' splicesite at nucleotide 653 and activating the common cryptic 3' splice siteat nucleotide 579 of intron 2. The efficiency of aberrant splicing ofIVS2⁶⁵⁴ pre-mRNA is higher than that for IVS2⁷⁰⁵ pre-mRNA and only smallamounts of correctly spliced product, relative to the aberrant one, aredetectable during splicing in vitro. In spite of the high efficiency ofaberrant splicing, oligo 3, targeted against the cryptic 3' splice site,as well as oligo 5, targeted against the aberrant 5' splice site,restored correct splicing efficiently at concentrations similar to thosedescribed above. At 2 μM concentration of either oligonucleotide thecorrectly spliced product accumulates and the aberrant product isvirtually undetectable (data not shown).

EXAMPLE 7 Reversal of Aberrant Splicing by Antisense OligonucleotideWhich Blocks the Human β-Globin Intron 1 Branch Point

This experiment is carried out essentially as described above to restorecorrect splicing in β-110 mutant pre-mRNA, except the oligonucleotidebinds to a sequence located just upstream from the native branch pointsequence of intron 1 of β-globin gene (nucleotides 75-88). The sequenceof the oligonucleotide is: CCCAAAGACUAUCC (SEQ ID NO:7). Correctsplicing is restored.

EXAMPLE 8 Construction of Cell Lines Expressing Thalassemic Humanβ-Globin pre-mRNA

A series of stable cell lines are constructed by transfecting HeLa cellsand CHO cells with thalassemic globin genes cloned under thecytomegalovirus (CMV) immediate early promoter. The genes includeIVS1-110, IVS2-654 and IVS1-5 mutation.

Stable cell lines are obtained in accordance with standard techniques.See, e.g., Current Protocols in Molecular Biology (P. Ausubel. et al.eds. 1987). Briefly, cells are cotransfected with plasmids carryingthalassemic globin genes under the CMV promoter and plasmids carryingthe neomycin resistance gene as a selectable marker (pSV2neo).Transfection is either by electroporation, (Z. Dominski and R. Kole,Mol. Cel. Biol. 11: 6075-6083 (1991); Z. Dominski and R. Kole, Mol.Cell. Biol. 12: 2108-2114 (1992)), or by the calcium phosphate method.Cells are plated and after 24-48 hours challenged with selective mediumcontaining G418. Surviving colonies are expanded and assayed forexpression of thalassemic globin mRNA as follows.

Total RNA is isolated from approximately 10⁵ cells using a commercialTri-Reagent (Molecular Research Center, Cincinnati, Ohio) followingmanufacturer's protocol. This method allows for easy processing of alarge number of small samples and gives high yields of good quality RNA.The splicing patterns are analyzed by RT-PCR using rTth polymerase andfollowing the manufacturers protocol (Perkin Elmer). No more than 1-5%of isolated RNA is required for detection of spliced RNA in transientlytransfected cells, thus the method is sufficiently sensitive for easydetection in stable cell lines. The reverse transcriptase step iscarried out with a 3' primer that hybridizes to the aberrant sequencesin thalassemic mRNA and spans the splice junction. This assures that thecontaminating DNA and normal globin RNA are not detected and do notinterfere with the assay. The cloned cell lines that express thalassemicpre-mRNA are used for treatment with antisense2'-O-methyl-oligonucleotides as described below.

EXAMPLE 9 Administration of Antisense Oligonucleotides In Cell Culture

Cells produced in Example 8 above are grown in 24 well culture dishescontaining 200 μl of media per well. 2×10⁴ cells are seeded per well andwhen attached they are treated with 200 μl of media containing up to 50μM concentration of antisense oligonucleotides. Cells are cultured up to4 days in the presence of the oligonucleotide before reaching confluence(2-3×10⁵ cells). Since 2'-O-methyl oligonucleotides are very stable inserum containing media, medium is changed no more than every two days.The 50 μM (40 μg per well) concentration of the oligonucleotiderepresents 100 fold excess over that required to elicit efficientrestoration of splicing in vitro. Even at this concentration a singleoligonucleotide synthesis at 1 μmole scale, yielding 1-1.6 mg of theoligonucleotide, provides sufficient material for 25 to 40 samples.

In an alternative approach cells are pretreated with Lipofectin™ reagent(DOTMA, from BRL) at a concentration of 10 μg/ml before addition ofoligonucleotides, in accordance with known techniques. (C. Bennett etal., Mol. Pharm. 41: 1023-1033 (1992)).

After treatment total RNA is isolated as above and assayed for thepresence of correctly spliced mRNA by RT-PCR. Amplification of primersis carried out in the presence of alpha-P32 labeled ATP to increasesensitivity of detection and reduce the number of cycles to 15.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GUCAGUGCCUAUCA14                                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AUAGACUAAUAGGC14                                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAUUAUUGCCCUGAAAG17                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCUCUUACCUCAGUUAC17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCUAUUACCUUAACCCAG18                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCUGACCACCAAC14                                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCCAAAGACUAUCC14                                                              __________________________________________________________________________

That which is claimed is:
 1. An oligonucleotide consisting of thenucleotide sequence shown as SEQ ID NO: 1, 3, 4, 5, or 7, whicholigonucleotide does not activate RNase H.
 2. An antisenseoligonucleotide according to claim 1, wherein said oligonucleotidecontains a modified internucleotide bridging phosphate residue selectedfrom the group consisting of methyl phosphonates, methylphosphonothioates, phosphoromorpholidates, phosphoropiperazidates, andphosphoramidates.
 3. An antisense oligonucleotide according to claim 1,wherein said antisense oligonucleotide contains a nucleotide having alower alkyl substituent at the 2' position thereof.
 4. An antisenseoligonucleotide according to claim 1 in a lipid vesicle.